Exceptions to the rule: case studies in the prediction of pathogenicity for genetic variants in hereditary cancer genes.

Based on current consensus guidelines and standard practice, many genetic variants detected in clinical testing are classified as disease causing based on their predicted impact on the normal expression or function of the gene in the absence of additional data. However, our laboratory has identified a subset of such variants in hereditary cancer genes for which compelling contradictory evidence emerged after the initial evaluation following the first observation of the variant. Three representative examples of variants in BRCA1, BRCA2 and MSH2 that are predicted to disrupt splicing, prematurely truncate the protein, or remove the start codon were evaluated for pathogenicity by analyzing clinical data with multiple classification algorithms. Available clinical data for all three variants contradicts the expected pathogenic classification. These variants illustrate potential pitfalls associated with standard approaches to variant classification as well as the challenges associated with monitoring data, updating classifications, and reporting potentially contradictory interpretations to the clinicians responsible for translating test outcomes to appropriate clinical action. It is important to address these challenges now as the model for clinical testing moves toward the use of large multi-gene panels and whole exome/genome analysis, which will dramatically increase the number of genetic variants identified.

Parental attitudes toward a diagnosis in children with unidentified multiple congenital anomaly syndromes.

One of the most common and unsatisfying situations encountered in medical genetics clinics is the child with multiple congenital anomalies (MCAs) suggestive of an underlying syndrome for whom it is not possible to make a definitive diagnosis. We undertook a qualitative, descriptive study to learn more about the ways in which the lack of a diagnosis affects parental coping and adjustment to their child's special needs. Semistructured interviews were conducted with 29 parents of 16 children born with an unidentified MCA syndrome. Interviews were based on a small number of open-ended questions, with follow-up probing, asking about parents' experiences with seeking a diagnosis, obtaining treatment and special services, explaining their child's problems to others, reproductive decision making, and support group participation. Transcripts of interviews were analyzed to identify the principal themes surrounding parents' beliefs about the significance of diagnostic information. The parents in this study had been aware of their child's anomalies for 2-23 years, and all had sought multiple evaluations to find a diagnosis. A majority of parents were still interested in identifying their child's syndrome, but most felt that their interest in a diagnosis had diminished over time, and some felt that there were benefits in not having this information. We identified six areas where parents claimed a diagnosis would have impact: labels, causes, prognosis, treatment, acceptance, and social support. Significant issues included obtaining special education services, anticipating the child's future and potential medical complications, life expectancy, recurrence risks, finding sources of social support, and ensuring that the child was receiving appropriate treatment. We conclude that the significance of diagnostic information is complex and varies for different parents. Providers should explore the underlying issues associated with a parental quest for a diagnosis in order to identify and address specific concerns. Published 2001 Wiley-Liss, Inc.

A protein similar to the 67 kDa laminin binding protein and p40 is probably a component of the translational machinery in Urechis caupo oocytes and embryos.

Oocytes of the echiuroid, Urechis caupo, contain an abundant maternal mRNA that encodes a protein very similar to LBP/p40, originally identified as a non-integrin 67 kDa laminin binding protein. We have sequenced the Urechis caupo mRNA for LBP/p40, and a similar mRNA from the Hawaiian sea urchin, Tripneustes gratilla. Both of the encoded proteins, as well as LBP/p40 proteins from other sources, share significant homology in the amino 2/3 of the proteins, but diverge extensively at the carboxyl ends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentration remains constant for the first 48 hours of embryogenesis and then begins to decline. In sucrose gradients run with homogenates from coelomocytes, oocytes, and early embryos, all of the LBP/p40 protein appears to be associated with either polysomes or free 40 S ribosomal subunits. In later embryos, an increasing proportion of the protein is found in the soluble fraction. Immunohistochemistry indicates that LBP/p40 is uniformly distributed in early U. caupo embryos, with no localization at the cell surface. In later embryos LBP/p40 is localized in specific parts of the embryo which may correspond to neural tissue.

Regulation of maternal messenger RNA translation during oogenesis and embryogenesis in Urechis caupo.

We have tested the hypothesis that the selective translation of mRNAs in oocytes and embryos is controlled by when during oogenesis individual maternal mRNAs are synthesized. In an earlier paper we described the isolation of cDNA probes to 21 maternal mRNAs which accumulate with different patterns during oogenesis in Urechis caupo (E. T. Rosenthal and F. H. Wilt, 1986, Dev. Biol. 117, 55-62). Many of these probes have now been used to analyze the translation of the maternal mRNAs in growing oocytes, full-grown oocytes, and embryos. The translation of all of the mRNAs studied changes dramatically when full-grown Urechis oocytes are fertilized. Our data do not support the idea that the translation of the different maternal mRNAs depends on when they were synthesized during oogenesis. This strongly suggests that, at some level, the ability of the cell's translational machinery to distinguish between different maternal mRNAs must reside directly, or indirectly, in the mRNA sequences. We have also found that in Urechis, as in Xenopus, the selective translation of maternal mRNAs is correlated with their selective adenylation. Consequently, we have sequenced the 3' ends of 16 translationally controlled maternal mRNAs in an effort to detect consensus sequences that might regulate adenylation. No such consensus sequences have been found, suggesting that the mRNA characteristics involved in the regulation of polyadenylation may reside in a wide variety of sequences or in more subtle features, such as secondary structure.

Widespread changes in the translation and adenylation of maternal messenger RNAs following fertilization of Spisula oocytes.

We have reported previously that sequence-specific adenylations and deadenylations accompany changes in the translation of maternal mRNA following fertilization of Spisula oocytes (E.T. Rosenthal, T.R. Tansey, and J.V. Ruderman, 1983, J. Mol. Biol. 166, 309-327). The data presented here confirm and extend those observations. We have identified four classes of maternal mRNA with respect to translation: Class 1-not translated in oocytes and translated at very high efficiency immediately after fertilization, Class 2-not translated in oocytes and partially utilized for translation following fertilization, Class 3-translated in oocytes and not translated in embryos, and Class 4-not translated either before or after fertilization. There is an excellent, although not perfect, correlation between the translation of an mRNA and its polyadenylation status. The poly(A) tails of all the mRNAs which are translated in oocytes and untranslated in embryos are shortened at fertilization, and the poly(A) tails of those mRNAs which are untranslated in oocytes and translated in embryos are lengthened at fertilization. These adenylations and deadenylations occur simultaneously during the first 20 min following fertilization.

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo.

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.

Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.

Cleavage in embryos of the sea urchin Arbacia punctulata consists of eight very rapid divisions that require continual protein synthesis to sustain them. This synthesis is programmed by stored maternal mRNAs, which code for three or four particularly abundant proteins whose synthesis is barely if at all detectable in the unfertilized egg. One of these proteins is destroyed every time the cells divide. Eggs of the sea urchin Lytechinus pictus and oocytes of the surf clam Spisula solidissima also contain proteins that only start to be made after fertilization and are destroyed at certain points in the cell division cycle. We propose to call these proteins the cyclins.

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes.

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.